Cd31shed as a molecular target for imaging of inflammation

ABSTRACT

Disclosed is CD31shed for use as a molecular imaging target in the molecular imaging of an inflammatory condition. Administering the radiolabeled peptide P8RI as CD31shed ligand in different rat models of inflammation indeed showed that CD31shed is present on activated cells in a quantity allowing a detectable signal, whereas the noise signal corresponding to CD31shed present on activated circulating cells and on other organs or cells not involved in inflammation was little. Also disclosed is a labeled CD31shed ligand and the use thereof as a molecular imaging agent in the molecular imaging of an inflammatory condition. The molecular imaging of inflammatory sites particularly allows determining whether a subject suffers from or is at risk of having an inflammatory condition or is at risk of recurrence of an inflammatory condition after an anti-inflammatory treatment.

FIELD OF THE INVENTION

The present invention relates to the molecular imaging of inflammatorysites, in particular for determining whether a subject suffers or is atrisk of having or developing an inflammatory condition and formonitoring the changes in the extent of an inflammatory condition afteran anti-inflammatory treatment.

BACKGROUND

Imaging of inflammation sites would be a useful tool for the diagnosisof an inflammation condition. The high level of glucose metabolism ininflammation has prompted to the use of ¹⁸F-FDG(2-deoxy-2-¹⁸F-fluoro-D-glucose) PET. ¹⁸F-FDG however suffers from poorspecificity and its high physiologic uptake in the heart, lung and brainlimits its use. More specific targets for imaging inflamed tissues aretherefore needed.

CD31 is a transmembrane glycoprotein receptor constitutively expressedby leukocytes, platelets and endothelial cells. It consists of a singlechain molecule comprising six Ig-like extracellular domains, a shorttransmembrane segment and a cytoplasmic tail comprising two importanttyrosine-based motifs (around Y663 and Y686) that act as ImmunoreceptorTyrosine-based Inhibitory Motifs (ITIMs). The structure of CD31 is shownin the Table 1 below.

TABLE 1 Structure of CD31 Domain Position on SEQ ID NO : 1 Signalpeptide  1 to 27 Extracellular domain  28 to 601 First Ig-likeextracellular domain  34 to 121 Second Ig-like extracellular domain 145to 233 Third Ig-like extracellular domain 236 to 315 Fourth Ig-likeextracellular domain 328 to 401 Fifth Ig-like extracellular domain 424to 493 Sixth Ig-like extracellular domain 499 to 591 Juxta-membranedomain 592 to 601 Transmembrane domain 602 to 620 Cytoplasmic domain 621to 738

Due to its homophilic and inhibitory functions, CD31 exerts a crucialrole in the homeostasis of the circulation. In pro-inflammatoryconditions, the homophilic portion of the receptor is lost due to acleavage and activated cells express a truncated form of CD31.WO2010/000756 indeed discloses that CD31 is shed on activated/memory Tlymphocytes between the 5th and the 6th extracellular Ig-like domainswhereas WO2013/152919 discloses that activated platelets also express aCD31 that is truncated between the 6^(th) extracellular domain and thejuxtamembrane sequence. Both types of shed extracellular sequences ofCD31 (referred to as “soluble CD31”) are then released into thecirculation, where they are present together with a soluble splicevariant of CD31, produced by healthy endothelial cells. The remainingsmall CD31 ectodomain which remains anchored to the membrane aftershedding is referred to as “CD31^(shed)”. WO2010/000756 andWO2013/152919 discloses a method for diagnosing a thrombotic or anautoimmune disorder based on the detection of said soluble CD31 in abiological sample of an individual.

WO2010/000741 discloses peptides corresponding to juxta-membrane aminoacids of the ectodomain of CD31 that are able to rescue thephysiological immunoregulatory function of CD31, by specificallytargeting CD31^(shed) on activated leukocytes and platelets. Suchpeptides are useful for the treatment of thrombotic or autoimmunedisorders. WO2013/190014 further discloses specific peptides of 8 aminoacids, within the membrane juxta-proximal part of extracellular CD31,which hold useful for the treatment of thrombotic or inflammatorydisorders and display physic-chemical properties that are more suitablefor drug development.

There is still a need to provide reliable solutions for the imaging anddiagnostic of inflammatory conditions.

DESCRIPTION OF THE INVENTION

The Inventors have unexpectedly found that CD31^(shed) itself can beused as a molecular target for the diagnosis of an inflammatorycondition, in particular by allowing the molecular imaging ofinflammatory sites. CD31^(shed) was indeed surprisingly found to bepresent on activated cells in a quantity allowing to obtain a detectablesignal when using a labeled CD31^(shed) ligand, whereas the noise signalcorresponding to CD31^(shed) present on activated circulating cells andon other organs or cells not involved in inflammation was little,thereby leading to a good signal to noise ratio. They have indeed shownin vivo in an animal model of inflammation that administering to saidanimal a radiolabeled CD31^(shed) ligand specific for CD31^(shed) allowslocalizing the inflammation site, due to the specific binding of theradiolabeled CD31^(shed) ligand to the activated cells expressingCD31^(shed) and its subsequent concentration in the inflammation site,whereas the remaining radiolabeled CD31^(shed) ligand is rapidly clearedfrom the body of the animal.

In this context, the Inventors used peptide P8RI of sequence SEQ ID NO:6 consisting of D-enantiomer amino acids coupled to HYNIC(6-Hydrazinopyridine-3-carboxylic acid) via a PEG spacer andradiolabeled with 99mTc. Said radiolabeled CD31^(shed) ligand was foundstable both in vitro and in vivo and specific to CD31^(shed), with verylow binding to plasma proteins.

Therefore, labelled CD31^(shed) ligands, in particular small peptides,able to specifically bind to CD31^(shed) are useful as a tracer formolecular imaging of inflammation.

One object of the present invention is thus a labeled CD31^(shed) ligandcomprising a CD31^(shed) ligand and at least one imaging label,preferably at least one radionuclide.

Said CD31^(shed) ligand is preferably:

-   -   a) a peptide selected in the group consisting of:        -   (i) a peptide consisting of a fragment of 3 to 15 amino            acids of the sequence defined by amino acids 579 to 601 of            sequence SEQ ID NO: 1,        -   (ii) a peptide consisting of a fragment of 3 to 15 amino            acids of a sequence corresponding to the amino acids 579 to            601 of sequence SEQ ID NO: 1 in a non-human mammalian CD31,        -   (iii) a peptide of 3 to 15 amino acids consisting of a            sequence at least 70% identical to the sequence of peptide            (i),        -   (iv) a peptide consisting of a retro-inverso sequence of            peptide (i), (ii) or (iii), and        -   (v) the peptide (i), (ii), (iii) or (iv) comprising at least            one or at least one further chemical modification,    -   or    -   b) a peptidomimetic of peptide a).

The CD31^(shed) ligand of (v) preferably comprises at least one aminoacid in the D-enantiomer form.

The CD31^(shed) ligand is preferably selected in the group consisting ofa peptide of sequence SEQ ID NO: 2, a peptide of sequence SEQ ID NO: 3,a peptide of sequence SEQ ID NO: 4, a peptide of sequence SEQ ID NO: 5,a peptide of sequence SEQ ID NO: 6 consisting of D-enantiomer aminoacids, a peptide of sequence SEQ ID NO: 7 and a peptide of sequence SEQID NO: 8 consisting of D-enantiomer amino acids.

A preferred CD31^(shed) ligand is a peptide of sequence SEQ ID NO: 5, ora peptide of sequence SEQ ID NO: 6 consisting of D-enantiomer aminoacids.

The radionuclide of the labeled CD31^(shed) ligand is preferablydetectable by molecular imaging technique(s), such as Positron EmissionTomography (PET), Single Photon Emission Computed Tomography (SPECT), anhybrid of SPECT and/or PET, or their combinations.

For example, the radionuclide is technetium-99m (99mTc or ^(99m)Tc),gallium-67 (⁶⁷Ga), gallium-68 (⁶⁸Ga), yttrium-90 (⁹⁰Y), indium-111(¹¹¹In), rhenium-186 (₁₈₆Re), fluorine-18 (¹⁸F), copper-64 (⁶⁴Cu) orthallium-201 (²⁰¹TI).

A preferred labeled CD31^(shed) ligand comprises a peptide of sequenceSEQ ID NO: 6 consisting of D-enantiomer amino acids as CD31^(shed)ligand and 99mTc as radionuclide.

In one preferred embodiment, the CD31^(shed) ligand is coupled withHYNIC (6-Hydrazinopyridine-3-carboxylic acid), optionally via at leastone spacer.

Another object of the present invention relates to the labeledCD31^(shed) ligand as defined above for use as a molecular imaging agentfor imaging of inflammatory sites, in particular for determining whethera subject suffers from an inflammatory condition, is at risk of havingan inflammatory condition or is at risk of recurrence of an inflammatorycondition after an anti-inflammatory treatment or for monitoring theefficacy of a treatment of an inflammatory condition. The presentinvention also relates to the use of the labeled CD31^(shed) ligand asdefined above as a molecular imaging agent, in particular for imaginginflammation sites.

Another object of the present invention relates to a method, preferablyan in vitro method, for determining whether a subject suffers from aninflammatory condition, is at risk of having an inflammatory conditionor is at risk of recurrence of an inflammatory condition after ananti-inflammatory treatment or for monitoring the efficacy of atreatment of an inflammatory condition, wherein said method comprises:

detecting the presence of CD31^(shed) on the surface of cells with alabeled CD31^(shed) ligand as defined above in a biological sampleobtained from the subject.

Another object of the present invention relates to CD31^(shed) for useas a molecular imaging target for imaging of inflammatory sites, moreparticularly for determining whether a subject suffers from aninflammatory condition, is at risk of having an inflammatory condition,is at risk of recurrence of an inflammatory condition after ananti-inflammatory treatment or for monitoring the efficacy of atreatment of an inflammatory condition. The present invention alsorelates to the use of CD31^(shed) for use as a molecular imaging target,in particular for imaging inflammation sites.

Inflammatory Condition

An inflammatory condition underlies a large number of diseases. Forexample, the immune system is often involved with inflammatoryconditions, as demonstrated in both allergic reactions and somemyopathies, with many immune system disorders resulting in abnormalinflammation.

As used throughout the present specification, the term “inflammatorycondition” includes but is not limited to a chronic inflammatorycondition, an immune disorder, an autoimmune disorder, an acute andchronic grant alloimmune conditions, an acute and chronicinfectious-driven inflammatory condition, a non-immune disease withetiological origins in inflammatory processes or their combinations.

The chronic inflammatory condition is for example inflammatory boweldisease, psoriasis, atopic dermatitis, cerebral amyloid angiopathy an/orvasculitis.

An immune disorder is for example allergies and/or myopathy.

The autoimmune disorder is for example rheumatoid arthritis (RA),multiple sclerosis (MS), inflammatory bowel disease (IBD), systemiclupus erythematodes (SLE), Graves' disease and/or diabetes mellitus.

Acute and chronic alloimmune conditions are for example allograftrejection or graft versus host disease (GVHD).

Acute and chronic infectious-driven inflammatory conditions include theformation of septic granulomas (abscess) and/or septic shock.

The non-immune disease with etiological origins in inflammatoryprocesses is for example cancer, thrombosis, ischaemic and/orischemia-reperfusion organ damage (for example heart and/or braininfarction) arterial inflammatory condition (such as atherothrombosis,arterial dissection and/or unhealed/thromboses arterial aneurysm) and/orneurodegenerative disease.

The inflammatory condition is for example selected from the groupconsisting of rheumatoid arthritis, multiple sclerosis, allergies,myopathy, inflammatory bowel disease, psoriasis, atopic dermatitis,cerebral amyloid angiopathy, vasculitis, systemic lupus erythematosus,Graves' disease, diabetes mellitus, acute or chronic graft rejection,cancer, thrombosis, atherothrombosis, ischaemic heart and/or braininfarction, and/or neurodegenerative disease.

Subject for Imaging, to be Diagnosed and/or Treated

A “subject” in the context of the present invention is a human being ora non-human mammal.

The terms “human being”, “individual” or “patient” are herein synonymousand may be used interchangeably.

Said human being may be of any sex, for example male or female and ofany age, for example an infant, child, adolescent, adult, elderlypeople.

A non-human mammal is preferably a mouse, rat, cat, dog, rabbit,hamster, swine, sheep, horse or primate.

The subject may suffer from an inflammatory condition, be suspected tosuffer from an inflammatory condition, be at risk of having aninflammatory condition or be at risk of recurrence of an inflammatorycondition.

The expressions “at risk of having an inflammatory condition”, “at riskof suffering from an inflammatory condition” and “at risks of developingan inflammatory condition” are herein synonymous.

“Risk” in the context of the present invention, relates to theprobability that an event will occur over a specific time period, forexample the conversion to an inflammatory condition.

Imaging of Inflammation

The expressions “imaging of inflammation sites” and “imaging ofinflammation” are herein synonymous.

Imaging of inflammation sites allows to localize the inflamed tissuesand/or inflamed organs in the body of a subject.

Imaging of inflammation sites presents many advantages.

For example, imaging of inflammation sites allows diagnosing aninflammation condition, confirming an inflammation condition, localizingthe inflamed tissues or organs, monitoring the response to a treatment,for example to an anti-inflammatory treatment, monitoring theinflammatory side-effects of a treatment, predicting the risk ofdeveloping an inflammatory condition and/or determining the risk ofrecurrence of an inflammatory condition after an anti-inflammatorytreatment.

Imaging of inflammation sites may thus be used for determining whether asubject suffers from an inflammatory condition, is at risk of having aninflammatory condition, is at risk of recurrence of an inflammatorycondition after an anti-inflammatory treatment or for monitoring theefficacy of a treatment of an inflammatory condition.

A molecular imaging target and/or a molecular imaging agent are neededto carry out imaging of inflammation.

As used herein, the term “molecular imaging target” refers to a compoundthat can be detected by using molecular imaging techniques.

As used herein, the term “molecular imaging agent” refers to a compoundthat can be used to detect specific biological elements, in particular amolecular imaging target, by using molecular imaging techniques.

The molecular imaging agent is preferably an agent coupled to an imaginglabel, covalently or non-covalently.

In the context of the invention, the molecular imaging agent is used todetect CD31^(shed) in vivo or in vitro, for example in a blood sample ofa subject.

CD31^(shed) for Use as a Molecular Imaging Target

The present invention thus relates to CD31^(shed) for use as a molecularimaging target for imaging of inflammation sites.

The present invention also relates to the use of CD31^(shed) as amolecular imaging target for imaging of inflammation sites.

The present invention particularly relates to CD31^(shed) for use as amolecular imaging target in a method for imaging inflammation sites,particularly in vivo.

As used herein “CD31^(shed)” refers to the remaining small CD31ectodomain which remains anchored to the membrane of endothelial cells,platelets or leukocytes, after shedding of the CD31 transmembraneglycoprotein receptor.

CD31^(shed) on the surface of activated endothelial cells, platelets andleukocytes lacks at least the 1^(st) to 5^(th) extracellular Ig-likedomains of CD31.

CD31^(shed) on the surface of activated platelets and activatedendothelial cells lacks the 1^(st) to 5^(th) extracellular Ig-likedomains of CD31 and at least one part of the 6th extracellular Ig-likedomain.

CD31^(shed) on the surface of activated leukocytes lacks the 1^(st) to5^(th) extracellular Ig-like domains, but comprises the 6thextracellular Ig-like domain.

The present invention also relates to CD31^(shed) for use as definedabove, in a method, preferably an in vivo method, for determiningwhether a subject suffers from an inflammatory condition, is at risk ofhaving an inflammatory condition, is at risk of recurrence of aninflammatory condition after an anti-inflammatory treatment or formonitoring the efficacy of a treatment of an inflammatory condition.

The present invention also relates to CD31^(shed) for use fordetermining whether a subject suffers from an inflammatory condition, isat risk of having an inflammatory condition, is at risk of recurrence ofan inflammatory condition after an anti-inflammatory treatment or formonitoring the efficacy of a treatment of an inflammatory condition, inparticular in vivo.

The expression “inflammatory condition” and “imaging of inflammationsites” are as defined above.

CD31^(shed) Ligand

The term “CD31^(shed) ligand” refers to any compound that is able tospecifically bind with CD31^(shed).

The specific binding of a compound to CD31^(shed) present on a cellsurface may be assessed by any method well-known by the skilled person.

For example, the specific binding may be measured by plasmon surfaceresonance, flow cytometry or beta-imager.

In a preferred embodiment, the binding of a compound to be tested toCD31^(shed) present on a cell surface is assessed as follows: thecompound to be tested, or an irrelevant analogue as negative control, isbound to a fluorescent probe (e.g. fluoresceine). The compound isincubated at consecutive dilutions (for example 1, 10, 100 μmol) withCD31+ cells (for example from a cell line, such as Jurkat T cells, orprimary cells, such as peripheral blood T cells), at a density of 10⁶cells/ml in a saline buffer comprising Ca++ and Mg++ (HBSS, culturemedium). Parallel conditions are incubated in the presence of a cellactivator (e.g. TCR crosslinking, such as 1 μg/ml anti-CD3eantibodies+20 μg/ml secondary F(ab′)2 fragment if the cells are Tlymphocytes). The reaction is stopped after 5 or 20 minutes by repeatedwashing steps with cold buffer. Cells are fixed with paraformaldehydeand washed again. Binding of the compound to be tested on individualcells is detected by the relative fluorescent signal using a flowcytometer. A greater signal for the specific compound as compared to thecontrol and in the conditions comprising the cell activator as comparedto resting cells indicates appropriate binding to CD31^(shed).

Alternatively, the compound to be tested, or an irrelevant analogue asnegative control, is tested for its binding to activated endothelialcells (for example primary cells from human blood vessels ofimmortalized cell lines). The cells are cultured on collagen type I thinlayer to confluence. For the activation, the cells are incubated with 20ng/ml Human recombinant TNFa for 30 minutes in culture medium. Thereaction is stopped by rinsing the cells with cold saline buffer and thecells are fixed (for instance with a Zinc-fixative solution for 10minutes at room temperature). After extensive rinsing in a saline buffercomprising, activated endothelial cells are labeled by the compound tobe tested, or an irrelevant analogue as negative control, is bound to afluorescent probe (e.g. fluoresceine) and counterstained by a plasmamembrane dye (e g Cell Mask™) and a nuclear staining (eg Hoechst 33342).Binding of the compound to be tested on individual cells is detected bythe relative fluorescent signal using a fluorescence microscope. Agreater signal for the specific compound as compared to the control andin the conditions comprising the cell activator as compared to restingcells indicates appropriate binding to CD31^(shed).

The CD31^(shed) ligand may be a peptide, a peptidomimetic, a chemicalcompound, an antibody or an aptamer.

The term “antibody” is thus used to refer to any antibody-like moleculethat has an antigen binding region, and this term includes antibodyfragments that comprise an antigen binding domain such as Fab′, Fab,F(ab′)2, single domain antibodies (DABs or VHH), TandAbs dimer, Fv, scFv(single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies,diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fabfusions, bispecific or trispecific, respectively); sc-diabody;kappa(lamda) bodies (scFv-CL fusions); DVD-Ig (dual variable domainantibody, bispecific format); SIP (small immunoprotein, a kind ofminibody); SMIP (“small modular immunopharmaceutical” scFv-Fc dimer;DART (ds-stabilized diabody “Dual Affinity ReTargeting”); small antibodymimetics comprising one or more CDRs and the like. The techniques forpreparing and using various antibody-based constructs and fragments arewell known in the art. In some embodiments, the antibody is a monoclonalantibody. In some embodiments, the antibody is non-internalizing. Asused herein the term “non-internalizing antibody” refers to an antibody,respectively, that has the property of to bind to a target antigenpresent on a cell surface, and that, when bound to its target antigen,does not enter the cell and become degraded in the lysosome. In someembodiments, the heterologous polypeptide is a light immunoglobulinchain. In some embodiments, the heterologous polypeptide is a heavyimmunoglobulin chain. In some embodiments, the heterologous polypeptideis a heavy single heavy chain variable domain of antibodies of the typethat can be found in Camelid mammals which are naturally devoid of lightchains.

Particularly, in the context of the invention, the antibody is a singledomain antibody. The term “single domain antibody” has its generalmeaning in the art and refers to the single heavy chain variable domainof antibodies of the type that can be found in Camelid mammals which arenaturally devoid of light chains. Such single domain antibody are alsocalled VHH or “nanobody®”. For a general description of (single) domainantibodies, reference is also made to the prior art cited above, as wellas to EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6),Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and WO 06/030220,WO 06/003388. The amino acid sequence and structure of a single domainantibody can be considered to be comprised of four framework regions or“FRs” which are referred to in the art and herein as “Framework region1” or “FRI”; as “Framework region 2” or “FR2”; as “Framework region 3”or “FR3”; and as “Framework region 4” or “FR4” respectively; whichframework regions are interrupted by three complementary determiningregions or “CDRs”, which are referred to in the art as “ComplementarityDetermining Region for “CDRI”; as “Complementarity Determining Region 1”or “CDR2” and as “Complementarity Determining Region 2” or “CDR3” and as“Complementarity Determining Region 2”, respectively. Accordingly, thesingle domain antibody can be defined as an amino acid sequence with thegeneral structure: FRI—CDRI—FR2—CDR2—FR3—CDR3—FR4 in which FRI to FR4refer to framework regions 1 to 4 respectively, and in which CDRI toCDR3 refer to the complementarity determining regions 1 to 3. In thecontext of the invention, the amino acid residues of the single domainantibody are numbered according to the general numbering for VH domainsgiven by the International ImMunoGeneTics information system aminoacidnumbering (http://imgt.cines.fr/).

Particularly, in the context of the invention, the antibody is a singlechain variable fragment. The term “single chain variable fragment” or“scFv fragment” refers to a single folded polypeptide comprising the VHand VL domains of an antibody linked through a linker molecule. In sucha scFv fragment, the VH and VL domains can be either in the VH—linker—VLor VL—linker—VH order. In addition to facilitate its production, a scFvfragment may contain a tag molecule linked to the scFv via a spacer. AscFv fragment thus comprises the VH and VL domains implicated intoantigen recognizing but not the immunogenic constant domains ofcorresponding antibody.

In one embodiment of the invention, the CD31^(shed) ligand is a peptide.

The peptide is preferably a peptide as disclosed in WO2010/000741 orWO2013/190014.

Preferably, the CD31^(shed) ligand is a synthetic peptide.

By a “synthetic peptide”, it is intended that the peptide is not presentwithin a living organism, e.g. within human body.

The synthetic peptide is preferably purified.

The synthetic peptide may be part of a composition or a kit.

The peptide may be selected in the group consisting of:

-   -   (i) a peptide consisting of a fragment of 3 to 15 amino acids of        the sequence defined by amino acids 579 to 601 of sequence SEQ        ID NO: 1,    -   (ii) a peptide consisting of a fragment of 3 to 15 amino acids        of a sequence corresponding to the amino acids 579 to 601 of        sequence SEQ ID NO: 1 in a non-human mammalian CD31,    -   (iii) a peptide of 3 to 15 amino acids consisting of a sequence        at least 70% identical to the sequence of peptide (i),    -   (iv) a peptide consisting of a retro-inverso sequence of peptide        (i), (ii) or (iii), and    -   (v) the peptide (i), (ii), (iii) or (iv) comprising at least one        or at least one further chemical modification, preferably at        least one amino acid in the D-enantiomer form.

A “fragment” refers herein to a sequence of consecutive amino acids. Forexample, a fragment may be a fragment of 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or 15 amino acids.

The sequence defined by amino acids 579 to 601 of sequence SEQ ID NO: 1is sequence SEQ ID NO: 12.

Thus, the peptide may consist of a fragment of 3 to 15 amino acids ofsequence SEQ ID NO: 12.

The peptide may also consist of a fragment of 3 to 15 amino acids of asequence corresponding to sequence SEQ ID NO: 12 in a non-humanmammalian CD31.

Non-limiting examples of non-human mammalian CD31 are the murine CD31 ofsequence SEQ ID NO: 9, the bovine CD31 of sequence SEQ ID NO: 10 and thepig CD31 of sequence SEQ ID NO: 11.

The person skilled in the art can easily identify a sequencecorresponding to the amino acids 579 to 601 of sequence SEQ ID NO: 1,i.e. to sequence SEQ ID NO: 12, in a non-human mammalian CD31 protein,for example by performing a sequence alignment between sequence SEQ IDNO: 1 and the sequence of said non-human mammalian CD31 protein, forexample with one of sequences SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO:11.

Methods for sequence alignment and determination of sequence identityare well known in the art, for example using publicly available computersoftware such as BioPerl, BLAST, BLAST-2, CS-BLAST, FASTA, ALIGN,ALIGN-2, LALIGN, Jaligner, matcher or Megalign (DNASTAR) software andalignment algorithms such as the Needleman-Wunsch and Smith-Watermanalgorithms.

The sequence of the CD31 peptide according to the invention ispreferably derived from the sequence of human CD31 or murine CD31.

The peptide may also be a peptide of 3 to 15 amino acids consisting of asequence at least 70%, at least 75%, at least 80%, at least 85% or atleast 90% identical to the sequence of peptide (i), i.e. to the sequenceof a fragment of 3 to 15 amino acids of the sequence defined by aminoacids 579 to 601 of sequence SEQ ID NO: 1.

A peptide sequence at least 70% identical to a given sequence of 4 to 6amino acids differs from said given sequence of at most one amino acid.A peptide sequence at least 70% identical to a given sequence of 7 to 9amino acids differs from said given sequence of at most two amino acids.

A peptide sequence at least 70% identical to a given sequence of 10 to13 amino acids differs from said given sequence of at most three aminoacids.

A peptide sequence at least 70% identical to a given sequence of 14 or15 amino acids differs from said given sequence of at most four aminoacids.

By “a sequence at least x % identical to a reference sequence”, it isintended that the amino acid sequence of the subject peptide isidentical to the reference sequence or differ from the referencesequence by up to 100-x amino acid alterations per each 100 amino acidsof the reference sequence. In other words, to obtain a polypeptidehaving an amino acid sequence at least x % identical to a referenceamino acid sequence, up to 100-x % of the amino acid residues in thesubject sequence may be inserted, deleted or substituted with anotheramino acid.

Methods for comparing the identity of two or more sequences are wellknown in the art. For instance, programs available in the WisconsinSequence Analysis Package, version 9.1, for example the programs BESTFITand GAP, may be used to determine the % identity between two polypeptidesequences. BESTFIT uses the “local homology” algorithm of Smith andWaterman and finds the best single region of similarity between twosequences. Other programs for determining identity between sequences arealso known in the art, for instance the Needle program, which is basedon the Needleman and Wunsch algorithm, described in Needleman and Wunsch(1970) J. Mol Biol. 48:443-453, with for example the followingparameters for polypeptide sequence comparison: comparison matrix:BLOSUM62, gap open penalty: 10 and gap extend penalty: 0.5, end gappenalty: false, end gap open penalty=10, end gap extend penalty=0.5; andthe following parameters for polynucleotide sequence comparison:comparison matrix: DNAFULL; gap open penalty=10, gap extend penalty=0.5,end gap penalty: false, end gap open penalty=10, end gap extendpenalty=0.5.

Peptides consisting of an amino acid sequence “at least 70%, 75%, 80%,85%, or 90% identical” to a reference sequence may comprise mutations,such as deletions, insertions and/or substitutions compared to thereference sequence.

In case of substitutions, the substitution preferably corresponds to aconservative substitution as indicated in the Table 1 below. In apreferred embodiment, the peptide consisting of an amino acid sequenceat least 70%, 75%, 80%, 85% or 90% identical to a reference sequenceonly differs from the reference sequence by conservative substitutions.

TABLE 1 Conservative substitutions Type of Amino Acid Ala, Val, Leu,Ile, Met, Pro, Amino acids with aliphatic hydrophobic Phe, Trp sidechains Ser, Tyr, Asn, Gln, Cys Amino acids with uncharged but polar sidechains Asp, Glu Amino acids with acidic side chains Lys, Arg, His Aminoacids with basic side chains Gly Neutral side chain

In another preferred embodiment, the peptide consisting of an amino acidsequence at least 70%, 75%, 80%, 85% or 90% identical to a referencesequence corresponds to a naturally-occurring allelic variant of thereference sequence.

In still another preferred embodiment, the peptide consisting of anamino acid sequence at least 70%, 75%, 80%, 85%or 90% identical to areference sequence corresponds to a homologous sequence derived fromanother non-human mammalian species than the reference sequence.

In a preferred embodiment, the peptide consisting of an amino acidsequence at least 70%, 75%, 80%, 85% or 90% identical to a referencesequence differs from the reference sequence by conservativesubstitutions and/or corresponds to a homologous sequence derived fromanother non-human mammalian species than the reference sequence.

By the expression “a peptide consisting of a retro-inverso sequence ofpeptide (i), (ii) or (iii)”, it is herein meant a peptide that differsfrom the peptide (i), (ii) or (iii) in that its amino acids are in thereverse order by comparison to the sequence of peptide (i), (ii) or(iii), respectively, and consist of D-amino acids instead of thenaturally-occurring L-amino acids.

D-enantiomers of amino acids (also called D-amino acids) are referred toby the same letter as their corresponding L-enantiomer (also calledL-amino acid), but in lower case. Thus, for example, the L-enantiomer ofarginine is referred to as ‘R’, while the D-enantiomer is referred to as‘r’.

Preferably, the peptide is soluble in an organic or nonorganic solvent.

In a preferred embodiment, the peptide is soluble in water. Moreparticularly, the peptide is preferably soluble in water and/or inaqueous buffer such as trifluoroacetate (for example in a 0.1%trifluoroacetate solution), NaCl 9 g/L, PBS, Tris or Tris-phosphate. Thesolubility in water and aqueous buffers is particularly advantageous onthe pharmacological point of view. Thanks to such solubility, thepeptide may be dissolved in an aqueous solution, for example at aconcentration equal to, at least of or at most of 1 micromolar, 10micromolar, 50 micromolar, 100 micromolar, 500 micromolar, 1 mM, 50 mMor 100 mM.

A CD31^(shed) ligand of the invention that is readily soluble in watermay be obtained by the presence of at least one charged amino acid(preferably arginine (R) and/or lysine (K)), wherein said charged aminoacid is not comprised between two hydrophobic residues.

Thus, in a preferred embodiment, the CD31^(shed) ligand according to theinvention comprises at least one charged amino acid, preferably arginineand/or lysine, wherein said charged amino acid is not comprised betweentwo hydrophobic residues.

In a more preferred embodiment, said charged amino acid is locatedeither at the N- or C-terminal end of the sequence.

For example, the sequence of a preferred CD31^(shed) ligand according tothe invention begins with the motif RV (for example instead of VRV).

In a preferred embodiment, the peptide is resistant to peptidase, inparticular to eukaryote peptidase.

By “resistant to peptidase”, it is herein meant that the CD31^(shed)ligand remains undigested, as determined by reversephase—high-performance liquid chromatography (RP-HPLC) and massspectroscopy (MS), upon incubation at 37° C. with mammalian serum orinjection in a living laboratory animal. Laboratory tests aimed atevaluating serum stability of the peptides are well standardized (seefor example Jenssen and Aspmo, 2008, Methods Mol Biol 494, 177-186).Highly peptidase-resistant peptides are those that remains undigestedfor up to 70% of their original mass and/or displaying a half lifelonger than 240 minutes in the presence of proteolytic enzymes (see forexample Kumarasinghe and Hruby, 2015, In Peptide Chemistry and DrugDesign, B. M. Dunn, ed. (Hoboken, N.J.: Wiley), pp. 247-266).

The CD31^(shed) ligand is preferably resistant to peptidases present inblood, such as soluble peptidases or peptidases present on cell surface.

The CD31^(shed) ligand may also comprise at least one or at least onefurther chemical modification, preferably to improve its stabilityand/or bioavailability.

Such chemical modifications generally aim at obtaining peptides withincreased protection of the peptides against enzymatic degradation invivo and/or increased capacity to cross membrane barriers, thusincreasing its half-life and/or maintaining or improving its biologicalactivity. Any chemical modification known in the art can be employedaccording to the present invention.

The CD31^(shed) ligand may comprise at least one artificial amino acid,said artificial amino acid being preferably selected from the groupconsisting of a D-enantiomer amino acid, a beta-methyl amino acid, aalpha-substituted alpha-amino acid and an amino acid analog.

By “beta-methyl amino acid”, it is herein meant a derivative of theamino acid alanine with an aminomethyl group on the side chain. Thisnon-proteinogenic amino acid is classified as a polar base.

By “alpha-substituted alpha-amino acid”, it is herein meant that thegroup on the alpha carbon of an L-amino acid (NH2) has been changed toanother, non proteinaceous group, such as a methyl-, aryl- or acyl-group.

By “amino acid analog”, it is herein meant any other artificial analogof a natural amino acid.

The CD31^(shed) ligand may thus comprise at least one amino acid in theD-enantiomer form. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15 of the amino acids of the CD31^(shed) ligand defined abovemay be in the D-enantiomer form.

In one embodiment, the CD31^(shed) ligand consists of D-amino acids.

The CD31^(shed) ligand may also comprise an inverted sequence, namely aninversion of the amino acid chain (from the C-terminal end to theN-terminal end). The entire amino acid sequence of the peptide may beinverted, or a portion of the amino acid sequence may be inverted. Forexample, a consecutive sequence of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15 amino acids may be inverted. Reference herein to ‘inverted’amino acids refers to inversion of the sequence of consecutive aminoacids in the sequence.

Other chemical modifications include, but are not limited to:

-   -   modifications to the N-terminal and/or C-terminal ends of the        peptides such as e.g. N-terminal methylation, N-terminal        acylation (preferably acetylation) or deamination, or        modification of the C-terminal carboxyl group into an amide or        an alcohol group;    -   modifications at the amide bond between two amino acids:        acylation (preferably acetylation) or alkylation (preferably        methylation) at the nitrogen atom or the alpha carbon of the        amide bond linking two amino acids;    -   modifications at the alpha carbon of the amide bond linking two        amino acids such as e.g. acylation (preferably acetylation) or        alkylation (preferably methylation) at the alpha carbon of the        amide bond linking two amino acids;    -   retro-inversions in which one or more naturally-occurring amino        acids (L-enantiomer) are replaced with the corresponding        D-enantiomers, together with an inversion of the amino acid        chain (from the C-terminal end to the N-terminal end);    -   azapeptides, in which one or more alpha carbons are replaced        with nitrogen atoms; betapeptides, in which the amino group of        one or more amino acid is bonded to the β carbon rather than the        a carbon,    -   ester linkage(s) (such as α-hydroxy acid(s)),    -   insertion of extra methylene group(s) (for example β- and        γ-amino acid(s), and/or    -   Peptoid(s), oligourea(s), arylamide(s) and/or oligohydrazide(s).

The CD31^(shed) ligand includes amino acids modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modification(s) can occur anywhere in a polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini, it will be appreciated that the same type of modification maybe present in the same or varying degrees at several sites in a givenpolypeptide. Also, a given polypeptide may contain many types ofmodifications. Polypeptides may be branched as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched and branched cyclic polypeptides may result fromnatural post-translational processes or may be made by syntheticmethods. Modifications include acetylation, acylation, ADP-ribosylation,araidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphatidyl inositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutarnate, formylation,gamma-carboxylation, glycosylation, GP6′I anchor formation,hydroxylation, iodination, mefhylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoyiation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

In a preferred embodiment of the invention, the CD31^(shed) ligand isselected in the group consisting of:

-   -   (i) a peptide consisting of a fragment of 3 to 15 amino acids of        the sequence defined by amino acids 579 to 601 of sequence SEQ        ID NO: 1, said fragment comprising the amino acids 579 to 581,        the amino acids 589 to 591, the amino acids 599 to 601 and/or        the amino acids 593 to 595 of SEQ ID NO: 1,    -   (ii) a peptide consisting of a fragment of 3 to 15 amino acids        of a sequence corresponding to the amino acids 579 to 601 of        sequence SEQ ID NO: 1 in a non-human mammalian CD31, for example        a fragment of 3 to 15 amino acids of the sequence defined by        amino acids 568 to 590 of sequence SEQ ID NO: 9, said fragment        preferably comprising the amino acids 568 to 570, the amino        acids 578 to 580, the amino acids 588 to 590 and/or the amino        acids 582 to 584 of SEQ ID NO: 9,    -   (iii) a peptide of 3 to 15 amino acids consisting of a sequence        at least 70% identical, preferably at least 75% identical,        preferably at least 80% identical, more preferably at least 85%        identical, still more preferably at least 90% identical to the        sequence of peptide (i),    -   (iv) a peptide consisting of a retro-inverso sequence of peptide        (i), (ii) or (iii), and    -   (v) the peptide (i), (ii), (iii) or (iv) comprising at least one        or at least one further chemical modification.

Such peptide has, for example, a sequence selected from the groupconsisting of: SSTLAVRVFLAPWKK (SEQ ID NO: 13, amino acids 576 to 590 ofSEQ ID NO: 9), STLAVRVFLAPWKK (SEQ ID NO: 14, amino acids 577 to 590 ofSEQ ID NO: 9), TLAVRVFLAPWKK (SEQ ID NO: 15, amino acids 578 to 590 ofSEQ ID NO: 9), LAVRVFLAPWKK (SEQ ID NO: 16, amino acids 579 to 590 ofSEQ ID NO: 9), AVRVFLAPWKK (SEQ ID NO: 17, amino acids 580 to 590 of SEQID NO: 9), VRVFLAPWKK (SEQ ID NO: 3, amino acids 581 to 590 of SEQ IDNO: 9), RVFLAPWKK (SEQ ID NO: 18, amino acids 582 to 590 of SEQ ID NO:9), VFLAPWKK (SEQ ID NO: 19, amino acids 583 to 590 of SEQ ID NO: 9),FLAPWKK (SEQ ID NO: 20, amino acids 584 to 590 of SEQ ID NO: 9), LAPWKK(SEQ ID NO: 2, amino acids 585 to 590 of SEQ ID NO: 9), APWKK (SEQ IDNO: 21, amino acids 586 to 590 of SEQ ID NO: 9), PWKK SEQ ID NO: 22,amino acids 587 to 590 of SEQ ID NO: 9), WKK (amino acids 588 to 590 ofSEQ ID NO: 9), SKILTVRVILAPWKK (SEQ ID NO: 23, amino acids 587 to 601 ofSEQ ID NO: 1), KILTVRVILAPWKK (SEQ ID NO: 24, amino acids 588 to 601 ofSEQ ID NO: 1), ILTVRVILAPWKK (SEQ ID NO: 25, amino acids 589 to 601 ofSEQ ID NO: 1), LTVRVILAPWKK (SEQ ID NO: 26, amino acids 590 to 601 ofSEQ ID NO: 1), TVRVILAPWKK (SEQ ID NO: 27, amino acids 591 to 601 of SEQID NO: 1), VRVILAPWKK (SEQ ID NO: 4, amino acids 592 to 601 of SEQ IDNO: 1), RVILAPWKK (SEQ ID NO: 28, amino acids 593 to 601 of SEQ ID NO:1), VILAPWKK (SEQ ID NO: 29, amino acids 594 to 601 of SEQ ID NO: 1),ILAPWKK (SEQ ID NO: 30, amino acids 595 to 601 of SEQ ID NO: 1),SSMRTSPRSSTLAVR (SEQ ID NO: 31, amino acids 568 to 582 of SEQ ID NO: 9),SSMRTSPRSSTLAV (SEQ ID NO: 32, amino acids 568 to 581 of SEQ ID NO: 9),SSMRTSPRSSTLA (SEQ ID NO: 33, amino acids 568 to 580 of SEQ ID NO: 9),SSMRTSPRSSTL (SEQ ID NO: 34, amino acids 568 to 579 of SEQ ID NO: 9),SSMRTSPRSST (SEQ ID NO: 35, amino acids 568 to 578 of SEQ ID NO: 9),SSMRTSPRSS (SEQ ID NO: 36, amino acids 568 to 577 of SEQ ID NO: 9),SSMRTSPRS (SEQ ID NO: 37, amino acids 568 to 576 of SEQ ID NO: 9),SSMRTSPR (SEQ ID NO: 38, amino acids 568 to 575 of SEQ ID NO: 9),SSMRTSP (SEQ ID NO: 39, amino acids 568 to 574 of SEQ ID NO: 9), SSMRTS(SEQ ID NO: 40, amino acids 568 to 573 of SEQ ID NO: 9), SSMRT (SEQ IDNO: 41, amino acids 568 to 572 of SEQ ID NO: 9), SSMR (SEQ ID NO: 42,amino acids 568 to 571 of SEQ ID NO: 9), SSM (amino acids 568 to 570 ofSEQ ID NO: 9), NHASSVPRSKILTVR (SEQ ID NO: 43, amino acids 579 to 593 ofSEQ ID NO: 1), NHASSVPRSKILTV (SEQ ID NO: 44, amino acids 579 to 592 ofSEQ ID NO: 1), NHASSVPRSKILT (SEQ ID NO: 45, amino acids 579 to 591 ofSEQ ID NO: 1), NHASSVPRSKIL (SEQ ID NO: 46, amino acids 579 to 590 ofSEQ ID NO: 1), NHASSVPRSKI (SEQ ID NO: 47, amino acids 579 to 589 of SEQID NO: 1), NHASSVPRSK (SEQ ID NO: 48, amino acids 579 to 588 of SEQ IDNO: 1), NHASSVPRS (SEQ ID NO: 49, amino acids 579 to 587 of SEQ ID NO:1), NHASSVPR (SEQ ID NO: 50, amino acids 579 to 586 of SEQ ID NO: 1),NHASSVP (SEQ ID NO: 51, amino acids 579 to 585 of SEQ ID NO: 1), NHASSV(SEQ ID NO: 52, amino acids 579 to 584 of SEQ ID NO: 1), NHASS (SEQ IDNO: 53, amino acids 579 to 583 of SEQ ID NO: 1), NHAS (SEQ ID NO: 54,amino acids 579 to 582 of SEQ ID NO: 1), NHA (amino acids 579 to 581 ofSEQ ID NO: 1), TSPRSSTLAVRVFLA (SEQ ID NO: 55, amino acids 572 to 586 ofSEQ ID NO: 9), SPRSSTLAVRVFL (SEQ ID NO: 56, amino acids 573 to 585 ofSEQ ID NO: 9), PRSSTLAVRVF (SEQ ID NO: 57, amino acids 574 to 584 of SEQID NO: 9), RSSTLAVRV (SEQ ID NO: 58, amino acids 575 to 583 of SEQ IDNO: 9), SSTLAVR (SEQ ID NO: 59, amino acids 576 to 582 of SEQ ID NO: 9),STLAV (SEQ ID NO: 60, amino acids 577 to 581 of SEQ ID NO: 9), TLA(amino acids 578 to 580 of SEQ ID NO: 9), SVPRSKILTVRVILA (SEQ ID NO:61, amino acids 583 to 597 of SEQ ID NO: 1), VPRSKILTVRVIL (SEQ ID NO:62, amino acids 584 to 596 of SEQ ID NO: 1), PRSKILTVRVI (SEQ ID NO: 63,amino acids 585 to 595 of SEQ ID NO: 1), RSKILTVRV (SEQ ID NO: 64, aminoacids 586 to 594 of SEQ ID NO: 1), SKILTVR (SEQ ID NO: 65, amino acids587 to 593 of SEQ ID NO: 1), KILTV (SEQ ID NO: 66, amino acids 588 to562 of SEQ ID NO: 1), ILT (amino acids 589 to 591 of SEQ ID NO: 1), RVF(amino acids 582 to 584 of SEQ ID NO: 9), RVFL (SEQ ID NO: 67, aminoacids 582 to 585 of SEQ ID NO: 9), RVFLA (SEQ ID NO: 68, amino acids 582to 586 of SEQ ID NO: 9), RVFLAP (SEQ ID NO: 69, amino acids 582 to 587of SEQ ID NO: 9), RVFLAPW (SEQ ID NO: 70, amino acids 582 to 588 of SEQID NO: 9), RVFLAPWK (SEQ ID NO: 5, amino acids 582 to 589 of SEQ ID NO:9), RVI (amino acids 593 to 595 of SEQ ID NO: 1), RVIL (SEQ ID NO: 71,amino acids 593 to 596 of SEQ ID NO: 1), RVILA (SEQ ID NO: 72, aminoacids 593 to 597 of SEQ ID NO: 1), RVILAP (SEQ ID NO: 73, amino acids593 to 598 of SEQ ID NO: 1), RVILAPW (SEQ ID NO: 74, amino acids 593 to599 of SEQ ID NO: 1), RVILAPWK (SEQ ID NO: 7, amino acids 593 to 600 ofSEQ ID NO: 1).

In a more preferred embodiment of the invention, the CD31^(shed) ligandis selected in the group consisting of:

-   -   (i) a peptide consisting of a fragment of 3 to 15 amino acids of        the sequence defined by amino acids 579 to 601 of sequence SEQ        ID NO: 1, said fragment comprising the amino acids 579 to 582,        the amino acids 588 to 592, the amino acids 598 to 601 and/or        the amino acids 593 to 595 of SEQ ID NO: 1,    -   (ii) a peptide consisting of a fragment of 3 to 15 amino acids        of a sequence corresponding to the amino acids 579 to 601 of        sequence SEQ ID NO: 1 in a non-human mammalian CD31, for example        a fragment of 3 to 15 amino acids of the sequence defined by        amino acids 568 to 590 of sequence SEQ ID NO: 9, said fragment        preferably comprising the amino acids 568 to 571, the amino        acids 578 to 580 the amino acids 587 to 590 and/or the amino        acids 582 to 584 of SEQ ID NO: 9,    -   (iii) a peptide of 3 to 15 amino acids consisting of a sequence        at least 70% identical, preferably at least 75% identical,        preferably at least 80% identical, more preferably at least 85%        identical, still more preferably at least 90% identical to the        sequence of peptide (i),    -   (iv) a peptide consisting of a retro-inverso sequence of peptide        (i), (ii) or (iii), and    -   (v) the peptide (i), (ii), (iii) or (iv) comprising at least one        or at least one further chemical modification.

In a preferred embodiment, the CD31^(shed) ligand is an 8 amino-acidfragment comprising inversions and/or at least one unnatural amino acid,such as at least one D-amino acids. Such peptides indeed retain theactivity of the original peptide or even demonstrate improved activity.Incorporation of unnatural amino acids in peptides intended fortherapeutic use is of utility in increasing the stability of thepeptide, in particular in vivo stability.

In another preferred embodiment of the invention, the CD31^(shed) ligandis selected in the group consisting of a peptide of sequence SEQ ID NO:2, a peptide of sequence SEQ ID NO: 3, a peptide of sequence SEQ ID NO:4, a peptide of sequence SEQ ID NO: 5, a peptide of sequence SEQ ID NO:6 consisting of D-enantiomer amino acids, a peptide of sequence SEQ IDNO: 7 and a peptide of sequence SEQ ID NO: 8 consisting of D-enantiomeramino acids.

A more preferred CD31^(shed) ligand is a peptide of sequence SEQ ID NO:5 or a peptide of sequence SEQ ID NO: 6 consisting of D-enantiomer aminoacids.

The CD31^(shed) ligand may additionally comprise at least one chelatingagent.

A chelating agent is a molecule covalently bound to the ligand, whichallows complexing radiometal(s).

Typically, chelating agents could be: 6-Hydrazinopyridine-3-carboxylicacid (HYNIC), chelating peptide such as Gly-Gly-Cys or His-basedsequence (Francesconi 2004, Waibel 1999, Ali 2011), MAG3 en N-ter(Okarvi 2012, 2004), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraceticacid (DOTA), diethylene triamine penta-acetic acid (DTPA),1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetylamide (DO3A),nitrilotriacetic acid (NTA), D-penicillamine, 2,3-dimercaptosuccinicacid,2,3-dimercapto-1-propanesulfonic acid, 2,3-dimercaptopropanol(BAL), triethylenetetramine, ammonium tetrathiomolybdate anion,ethylenediaminetetraacetic acid (EDTA),2-(p-isothiocyanatobenzyl)-6-methyldiethylenetriaminepentaacetic acid(IB4M) or hydroxypyridinone (HOPO).

The CD31^(shed) ligand may be linked to the chelating agent directly(for example the chelating agent being attached to a lateral amino acidlong chain) or indirectly, for example via at least one spacer.

A preferred chelating agent is 6-Hydrazinopyridine-3-carboxylic acid(HYNIC).

In such case, the peptide of the CD31^(shed) ligand may be linked toHYNIC directly (for example HYNIC being attached to a lateral amino acidlong chain) or indirectly, for example via at least one spacer.

The spacer for example comprises or consists of at least one PEG(polyethylene glycol), for example one, two or at least three PEG, morepreferably three PEG and/or at least one aliphatic spacer.

A preferred CD31^(shed) ligand is a compound of the following formula:

This compound is also called HYNIC-P8RI in the following.

The CD31^(shed) ligand may be prepared by any well-known procedure inthe art, such as chemical synthesis, for example solid phase synthesisor liquid phase synthesis, or genetic engineering. As a solid phasesynthesis, for example, the amino acid corresponding to the C-terminusof the peptide to be synthesized is bound to a support which isinsoluble in organic solvents, and by alternate repetition of reactions,one wherein amino acids with their amino groups and side chainfunctional groups protected with appropriate protective groups arecondensed one by one in order from the C-terminus to the N-terminus, andone where the amino acids bound to the resin or the protective group ofthe amino groups of the peptides are released, the peptide chain is thusextended in this manner. After synthesis of the desired peptide, it issubjected to the deprotection reaction and cut out from the solidsupport. Such peptide cutting reaction may be carried with hydrogenfluoride or tri-fluoromethane sulfonic acid for the Boc method, and withTFA for the Fmoc method.

Solid phase synthesis methods are largely classified by the tBoc methodand the Fmoc method, depending on the type of protective group used.Typically used protective groups include tBoe (t-butoxycarbonyl), CI-Z(2-chlorobenzyloxycarbonyl), Br-Z (2-bromobenzyloyycarbonyl), Bzl(benzyl), Fmoc (9-fluorenylmcthoxycarbonyl), Mbh(4,4′-dimethoxydibenzhydryl), Mtr(4-methoxy-2,3,6-trimethylbenzenesulphonyl), Trt (trityl), Tos (tosyl),Z (benzyloxycarbonyl) and Clz-Bzl (2,6-dichlorobenzyl) for the aminogroups; NO2 (nitro) and Pmc (2,2, 5,7,8-pentamethylchromane-6-sulphonyl) for the guanidino groups); and tBu(t-butyl) for the hydroxyl groups).

Alternatively, the CD31^(shed) ligand may be synthesized usingrecombinant techniques.

The method of producing the CD31^(shed) ligand may optionally comprisethe steps of purifying said CD31^(shed) ligand, chemically modifyingsaid CD31^(shed) ligand, and/or formulating said CD31^(shed) ligand intoa pharmaceutical composition.

In an embodiment, the CD31^(shed) ligand is a peptidomimetic of apeptide as defined above, i.e. a compound that mimics said peptide.

A «peptidomimetic» is a compound consisting of or essentially consistingof non-peptidic structural elements that mimics a given peptide, therebyconferring to said compound a biological activity equal to or similar tosaid peptide.

The peptidomimetic is preferably soluble in an organic or nonorganicsolvent.

As the peptide previously, the peptidomimetic is preferably soluble inwater.

Methods for designing and synthesizing peptidomimetics of a givenpeptide are well-known in the art and include e.g. those described inRipka and Rich (Curr. Opin. Chem. Biol. 1998; 2(4):441-52) and in Patchand Barron (Curr. Opin. Chem. Biol. 2002; 6(6):872-7).

Imaging Label

The labeled CD31^(shed) ligand according to the invention comprises atleast one imaging label.

Said imaging label may be a radionucleide or a contrastophor.

The term “contrastophor” as used herein refers to a contrast agent, forexample comprising a chelating agent.

Examples of chelating agent that may be used in a contrastophor aregiven below in the section “CD31^(shed) ligand”.

Non-limitative examples of contrastophor are Gd-DTPA (complex ofgadolinium and DTPA) or Gd-DOTA (complex of gadolinium and DOTA).

A contrastophor is preferably used in MRI (also called thereafter “IRM”or “Magnetic Resonance Imaging”).

The term “radionuclide” as used herein has the same meaning asradioactive nuclide, radioisotope or radioactive isotope.

The radionuclide is preferably detectable by nuclear medicine molecularimaging technique(s), such as, Positron Emission Tomography (PET),Single Photon Emission Computed Tomography (SPECT), an hybrid of SPECTand/or PET or their combinations.

Single Photon Emission Computed Tomography (SPECT) herein includesplanar scintigraphy (PS).

An hybrid of SPECT and/or PET is for example SPECT/CT, PET/CT, PET/IRMor SPECT/IRM.

SPECT and PET acquire information on the concentration (or uptake) ofradionuclides introduced into a subject's body. PET generates images bydetecting pairs of gamma rays emitted indirectly by a positron-emittingradionuclide. A PET analysis results in a series of thin slice images ofthe body over the region of interest (e.g., brain, breast, liver, . . .). These thin slice images can be assembled into a three dimensionalrepresentation of the examined area. SPECT is similar to PET, but theradioactive substances used in SPECT have longer decay times than thoseused in PET and emit single instead of double gamma rays. Although SPECTimages exhibit less sensitivity and are less detailed than PET images,the SPECT technique is much less expensive than PET and offers theadvantage of not requiring the proximity of a particle accelerator.Actual clinical PET presents higher sensitivity and better spatialresolution than SPECT, and presents the advantage of an accurateattenuation correction due to the high energy of photons; so PETprovides more accurate quantitative data than SPECT. Planar scintigraphy(PS) is similar to SPECT in that it uses the same radionuclides.However, PS only generates 2D-information.

SPECT produces computer-generated images of local radiotracer uptake,while CT produces 3-D anatomic images of X ray density of the humanbody. Combined SPECT/CT imaging provides sequentially functionalinformation from SPECT and the anatomic information from CT, obtainedduring a single examination. CT data are also used for rapid and optimalattenuation correction of the single photon emission data. By preciselylocalizing areas of abnormal and/or physiological tracer uptake,SPECT/CT improves sensitivity and specificity, but can also aid inachieving accurate dosimetric estimates as well as in guidinginterventional procedures or in better defining the target volume forexternal beam radiation therapy. Gamma camera imaging with single photonemitting radiotracers represents the majority of procedures.

In a preferred embodiment, the radionuclide is detectable by SPECT or anhybrid SPECT/CT.

The radionuclide may be selected in the group consisting oftechnetium-99m (^(99m)Tc), gallium-67 (⁶⁷Ga), gallium-68 (⁶⁸Ga)yttrium-90 (⁹⁰Y), indium-111 (¹¹¹In), rhenium-186 (¹⁸⁶RE), flourine-18(¹⁸F), copper-64 (⁶⁴Cu) or thallium-201 (²⁰¹TI).

A preferred radionuclide is technetium-99m (99mTc).

Labeled CD31^(shed) Ligand

The present invention thus particularly relates to a labeled CD31^(shed)ligand comprising a CD31^(shed) ligand and at least one imaging label.

The CD31^(shed) ligand and the imaging label are as defined above in thesections of the same name.

The radiochemical specific activity of labeled CD31^(shed) ligand ispreferably greater than 70 GBq/pmol, more preferably greater than 80GBq/pmol, more preferably greater than 90 GBq/pmol, for example greaterthan 100 GBq/pmol.

The labeled CD31^(shed) ligand is preferably stable.

The stability of the labeled CD31^(shed) ligand may be assessed in vitroand/or in vivo by any method well known by the skilled person, forexample by determining radiochemical purity (RCP).

The radiochemical purity (RCP) may be assessed by high performanceliquid chromatography (HPLC) and/or instant thin layer chromatography(ITLC), preferably using both methods.

For example, the in vitro stability may be assessed after incubation ofthe labeled CD31^(shed) ligand in vitro in plasma, preferably in humanplasma at 37° C., for example for at least 1 hour, at least 2 hours, atleast 3 hours or at least 4 hours.

For example, the in vivo stability may be assessed in a sample of humanor non-human mammal urine, for example of rat urine, afteradministration of the labeled CD31^(shed) ligand in said human ornon-human mammal.

The in vitro and/or in vivo RCP of the labeled CD31^(shed) ligand ispreferably greater than 80%, preferably greater than 85%, for examplegreater than 89%.

The biodistribution and specific uptake of the labeled CD31^(shed)ligand by activated platelets, endothelial cells and leukocytes may beassessed in an inflammation model, for example in a rat model of heartinflammation, brain inflammation or vascular inflammation, such as theexperimental abdominal aorta aneurysm (AAA) induced by local infusion ofelastase and followed by intravenous injection of a periodontalbacterium present in human AAA and known to induce AAA inflammation. Forexample, immediately after intravenous injection of the labeledCD31^(shed) ligand, sequential whole-body acquisitions (for exampleevery 10 minutes for the first hour) are performed, for example using ahybrid SPECT/CT camera (NanoSPECT/CT, Bioscan Inc.).

The binding of the labeled CD31^(shed) ligand to plasma proteins ispreferably low, for example lower than 10%, more preferably lower than5% after 4 hours of incubation in human plasma at 37° C.

The low binding to plasma proteins may be assessed by any method wellknown by the skilled person, such as size exclusion chromatography, forexample after incubation of the labeled CD31^(shed) ligand in vitro inplasma, preferably in human plasma, at 37° C., for example for at least1 hour, at least 2 hours, at least 3 hours or at least 4 hours.

The CD31^(shed) ligand and the imaging label may be linked covalently ornon-covalently.

The CD31^(shed) ligand and the imaging label are preferably linkednon-covalently.

When the CD31^(shed) ligand comprises a chelating agent, the CD31^(shed)ligand is preferably linked to the imaging label via said chelatingagent, and preferably non covalently.

The labeled CD31^(shed) ligand is preferably a radiolabeled CD31^(shed)ligand.

A preferred labeled CD31^(shed) ligand thus comprises a CD31^(shed)ligand and at least one radionuclide.

In one preferred embodiment of the invention, the labeled CD31^(shed)ligand comprises a peptide of sequence SEQ ID NO: 6 consisting ofD-enantiomer amino acids as CD31^(shed) ligand and ⁹⁹mTc as aradionuclide.

The labeled CD31^(shed) ligand may additionally comprise at least oneco-ligand, i.e. one or more ligands coupled to the CD31^(shed) ligand,

The co-ligand(s) is/are preferably used to stabilize the imaging labelbinding to the CD31^(shed) ligand.

For example, the co-ligand may be tricine, EDDA (ethylendiaminediaceticacid) (EDDA), aminocarboxylate, phosphine or pyridine.

In on embodiment, the labeled CD31^(shed) ligand comprises both tricineand EDDA as co-ligands, for example when the radionuclide is 99mTc.

When at least one co-ligand is used, the labeled CD31^(shed) ligandcomprises or consists of a complex comprising said co-ligand(s), saidCD31^(shed) ligand and said radionuclide.

In one embodiment of the invention, the CD31^(shed) ligand is thepeptide of sequence SEQ ID NO: 6 consisting of D-enantiomer amino acidscoupled with HYNIC via a spacer as defined above.

A preferred labeled CD31^(shed) ligand, referred to as 99mTc-HYNIC-P8RI,comprises:

the compound of formula:

and

the radionuclide 99mTc.

HYNIC forms a 99mTc-N bond between technetium and the hydrazine moietyof HYNIC.

The labeled CD31^(shed) ligand can be prepared by conventional methodsperfectly known by a man skilled in the art.

In the methods and uses described herein, the labeled CD31^(shed) ligandmay be used per se or as a pharmaceutical composition as defined below.

The methods and uses described herein may be performed by using a kit asdefined below.

Labeled CD31^(shed) Ligand for Use as a Molecular Imaging Agent

The present invention also relates to the use of a labeled CD31^(shed)ligand as defined above as a molecular imaging agent for imaginginflammation sites.

The present invention thus also relates to a labeled CD31^(shed) ligandas defined above for use as a molecular imaging agent for imaging ofinflammatory sites.

The present invention particularly relates to a labeled CD31^(shed)ligand as defined above for use as a molecular imaging agent in an invivo method of detecting, more particularly imaging, inflammatory sites.

In particular, the present invention relates to a labeled CD31^(shed)ligand for use as defined above, in a method, preferably an in vivomethod, for determining whether a subject suffers from an inflammatorycondition, is at risk of having an inflammatory condition or is at riskof recurrence of an inflammatory condition after an anti-inflammatorytreatment or for monitoring the efficacy of a treatment of aninflammatory condition.

The invention also relates to a labeled CD31^(shed) ligand for use asdefined above, wherein the presence, localization and/or amount ofCD31^(shed) is determined. The labeled CD31^(shed) ligand indeed bindsto CD31^(shed).

The presence, localization and/or amount of CD31^(shed) is determined bythe presence, localization and/or amount of the labeled CD31^(shed)ligand, i.e. of the signal detected corresponding to the imaging labelof said labeled CD31^(shed) ligand.

The presence, localization and/or amount of the labeled CD31^(shed)ligand may be determined by planar scintigraphy (PS), Positron EmissionTomography (PET), Single Photon Emission Computed Tomography (SPECT), anhybrid SPECT/CT or their combinations.

The labeled CD31^(shed) ligand is preferably used or administeredintravenously.

The labeled CD31^(shed) ligand is preferably used or administered in anamount sufficient to obtain a detectable signal, in particular oneinjected intravenously.

The signal corresponding to the imaging label of the labeled CD31^(shed)ligand is preferably detected immediately after the administration ofthe labeled CD31^(shed) ligand.

The signal may be detected in the entire body of the subject or in onlyone part of the subject (particularly in only one part of the body ofthe subject).

The present invention thus particularly relates to a labeled CD31^(shed)ligand as defined above for use in a method for detecting, moreparticularly imaging, inflammation sites, wherein said method comprises:

-   -   administering said labeled CD31^(shed) ligand to the subject,        and    -   imaging said subject or at least one part of the body of said        subject, thereby detecting the binding of the labeled        CD31^(shed) ligand to CD31^(shed) in said subject or in said        part of the body of the subject.

The present invention thus particularly relates to a labeled CD31^(shed)ligand as defined above for use in a method for determining whether asubject suffers from an inflammatory condition, is at risk of having aninflammatory condition or is at risk of recurrence of an inflammatorycondition after an anti-inflammatory treatment or for monitoring theefficacy of a treatment of an inflammatory condition, wherein saidmethod comprises:

-   -   administering said labeled CD31^(shed) ligand to the subject,        and    -   imaging said subject or at least one part of the body of said        subject, thereby detecting the binding of the labeled        CD31^(shed) ligand to CD31^(shed) in said subject or in said        part of the body of the subject.

The molecular imaging agent of the invention represents a powerful toolfor diagnosing or assessing an inflammatory condition associated withCD31^(shed).

-   -   a) Determining if a subject suffers from an inflammatory or is        at risk of suffering from an inflammatory condition

In a method for determining whether a subject suffers from aninflammatory condition or is at risk of suffering from an inflammatorycondition, the subject to be diagnosed is suspected to suffer or belikely to suffer from an inflammatory condition or suffers from aninflammatory condition. The method can indeed be performed to confirmthat the subject suffers from an inflammatory condition and/or tospecify the kind of inflammatory condition.

The presence, localization and/or amount of CD31^(shed) may indicatethat the subject suffers from an inflammatory condition.

The localization of CD31^(shed) may further indicate the kind ofinflammatory condition.

The amount of CD31^(shed) may further indicate the severity of theinflammation condition.

The presence, localization and/or amount of CD31^(shed) may be comparedto a reference value or a control biological image. When the referencevalue or the control biological sample corresponds to a healthy subject,in particular a subject that does not suffer from an inflammatorycondition or a panel of healthy subjects, a presence, localizationand/or amount of CD31^(shed) detected in the biological sample that isgreater than those of reference value or of the control image indicatesthat the subject suffers or, is at risk of having an inflammatorycondition.

-   -   b) Determining the risk of recurrence of an inflammatory        condition after an anti-inflammatory treatment

The methods and uses according to the invention can also be used tofollow the risk of recurrence of an inflammatory condition after ananti-inflammatory treatment. In particular, they can be used to monitorthe subject after an anti-inflammatory treatment. For example, this canbe achieved by repeating the method at least one time, preferably twotimes after the end of said treatment, in order to determine thepresence, localization and/or amount of CD31^(shed). An increasedpresence, localization and/or amount of CD31^(shed) may indicate a highrisk of recurrence of an inflammatory condition after ananti-inflammatory treatment. A stable or a decreased presence,localization and/or amount of CD31^(shed) may indicate a low risk ofrecurrence of an inflammatory condition after an anti-inflammatorytreatment. For example, an image of a biological sample and/or of thesubject or at least one part of the subject is generated at the end ofthe treatment and at least one time after treatment. Comparison of theimages at the end and after treatment allows the risk of recurrence ofan inflammatory condition after an anti-inflammatory treatment to bemonitored.

-   -   c) Monitoring the response to a treatment of a subject suffering        from an inflammatory condition

The methods and uses according to the invention can also be used tomonitor the response to a treatment of a subject suffering from aninflammatory condition. For example, this can be achieved by repeatingthe method at least two times, for example one time before treatment andat least two times during the treatment, or at least two times duringthe treatment. For example, an image of a biological sample and/or ofthe subject or at least one part of the subject is generated beforetreatment and at least one time during the treatment, for example withan anti-inflammatory treatment. Comparison of the images before andduring treatment allows the response of the subject to that particulartreatment to be monitored.

An increased presence, localization and/or amount of CD31^(shed) mayindicate that the treatment is not or not any more efficient. A stableor a decreased presence, localization and/or amount of CD31^(shed) mayindicate that the treatment is efficient.

The expression “monitoring the response to a treatment” and “monitoringthe efficacy of a treatment” are herein synonymous.

Said treatment may be a curative or preventive treatment.

Said treatment may comprise or consist in administering at least oneanti-inflammatory agent, at least one immunosuppressant, at least oneprobiotic (i.e. live microorganisms that may confer a health benefit onthe subject), at least one antibiotic or their combinations.

Method Using a Labeled CD31^(shed) Ligand as a Molecular Imaging Agent

The present invention also relates to a method for imaging ofinflammation sites using a labeled CD31^(shed) ligand as defined above.

The present invention also relates to a method, preferably an in vitromethod, for determining whether a subject suffers from an inflammatorycondition, is at risk of having an inflammatory condition or is at riskof recurrence of an inflammatory condition after an anti-inflammatorytreatment or for monitoring the efficacy of a treatment of aninflammatory condition in a subject, wherein said method comprisesdetecting the presence of CD31^(shed) on the surface of cells with alabeled CD31^(shed) ligand as defined above in a biological sample ofthe subject.

The cells are preferably platelets, leukocytes and/or endothelial cells.

By “biological sample”, it is herein meant any sample able to containendothelial cells, platelets or leukocytes, such as a blood sample orfraction thereof (for example a thrombus) or a tissue sample, forexample a biopsy.

The method may comprise a first step of providing a biological samplefrom the subject.

The present invention also relates to a method as defined above, whereinthe detection of CD31^(shed) on the surface of the cells in saidbiological sample indicates that the subject suffers, is at risk ofhaving or developing an inflammatory condition or is at risk ofrecurrence of an inflammatory condition.

The present invention also relates to a method as defined above, whereinthe amount of CD31^(shed) detected on the surface of the cells in saidbiological sample is compared to a reference value or a controlbiological sample. When the reference value or the control biologicalsample corresponds to a healthy subject, in particular a subject thatdoes not suffer from an inflammatory condition or a panel of healthysubjects, an amount of CD31^(shed) detected in the biological samplethat is greater than those of reference value or of the controlbiological sample indicates that the subject suffers, is at risk ofhaving or developing an inflammatory condition or is at risk ofrecurrence of an inflammatory condition.

The present invention also relates to a method as defined above, whereinan increased amount of CD31^(shed) detected on the surface of the cellsin said biological sample by comparison to the amount detected in abiological sample before treatment or earlier during treatment indicatesthat said treatment is not efficient and/or wherein a stable or adecreased amount of CD31^(shed) detected on the surface of the cells insaid biological sample by comparison to the amount detected in abiological sample before treatment or earlier during treatment indicatesthat said treatment is efficient.

Said method may further comprise determining the amount of CD31^(shed)on the surface of cells.

The detection of CD31^(shed) on the surface of the cells in saidbiological sample is preferably carried out by imaging techniques.

The step of detection as mentioned above comprises contacting thebiological sample of the subject with a labeled CD31^(shed) ligand,preferably an effective amount of a labeled CD31^(shed) ligand, as amolecular imaging agent.

The present invention also relates to a method as defined above, whereinsaid method comprises:

-   -   contacting a biological sample of the subject with a labeled        CD31^(shed) ligand, and    -   detecting the labeled CD31^(shed) ligand bound to CD31^(shed),        thereby detecting the presence of CD31^(shed) on the surface of        cells in the biological sample.

The contact is preferably carried out under conditions that allow themolecular imaging agent (1) to reach the cells of the subject that mayexpress CD31^(shed) (preferably endothelial cells, platelets and/orleukocyte) and (2) to interact with such CD31^(shed), so that theinteraction results in the binding of the molecular imaging agent to theCD31^(shed). After contact with the labeled CD31^(shed) ligand and aftersufficient time has elapsed for the interaction to take place, themolecular imaging agent bound to CD31^(shed) present in the subjectsample is detected by a molecular imaging technique as disclosed above.

An “effective amount” is an amount sufficient to allow the molecularimaging agent to complete these three conditions of (1) reaching thecells, (2) interacting with CD31^(shed) and (3) being detected.

Method of Treatment of an Inflammation Condition

The present invention also relates to a method for preventing and/ortreating an inflammatory condition in a subject in need thereof, whereinsaid method comprises,

-   -   performing a method as defined above for determining whether a        subject suffers from an inflammatory condition, is at risk of        having an inflammatory condition or is at risk of recurrence of        an inflammatory condition after an anti-inflammatory treatment        or for monitoring the efficacy of a treatment of an inflammatory        condition in a subject,    -   when the subject is determined to suffer from an inflammatory        condition, to be at risk of having an inflammatory condition or        to be at risk of recurrence of an inflammatory condition after        an anti-inflammatory treatment or if the treatment is not        efficient, administering to said subject a suitable treatment.

Said suitable treatment may comprise or consist in administering atleast one anti-inflammatory agent, at least one immunosuppressant, atleast one probiotic (i.e. live microorganisms that may confer a healthbenefit on the subject), at least one antibiotic, at least oneCD31^(shed) ligand linked to an active ingredient or their combinations.

The present invention also relates to a method for preventing and/ortreating an inflammatory condition in a subject in need thereof, whereinsaid method comprises administering to said subject a CD31^(shed) ligandlinked to an active ingredient.

The active ingredient is for example a drug, such as ananti-inflammatory agent. The CD31^(shed) ligand is thereby used as adrug targeting agent and allows improving the effect of said drug.

Pharmaceutical Composition Comprising the Molecular Imaging Agent

The pharmaceutical composition according to the invention comprises atleast one labeled CD31^(shed) ligand and at least one pharmaceuticallyacceptable carrier.

As used herein, the term “pharmaceutically acceptable carrier” refers toa carrier medium which does not interfere with the effectiveness of thebiological activity of the active ingredient, i.e. the molecular imagingagent, and which is not excessively toxic to the subject at theconcentration(s) at which it is administered. This term includessolvent(s), dispersion medium/media, coating(s), antibacterial and/orantifungal agent(s), isotonic agent(s), adsorption delaying agent(s) andtheir combinations. The use of such medium/media and/or agent(s) forpharmaceutically active substance(s) is well known in the art.

The pharmaceutical composition may be administered by injection. Foradministration by injection, the pharmaceutical composition comprisingthe molecular imaging agent may be formulated as sterile aqueous ornon-aqueous solution or alternatively as sterile powder for theextemporaneous preparation of a sterile injectable solution. Thepharmaceutical composition should be stable under the conditions ofmanufacture and storage, and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi.

Pharmaceutically acceptable carrier(s) for administration by injectionare solvent(s) or dispersion medium/media, such as aqueous solution(s)(e.g., Hank's solution, alcoholic/aqueous solution or saline solution),and non-aqueous carrier(s) (e.g. propylene glycol, polyethylene glycol,vegetable oil and injectable organic esters such as ethyl oleate). Theinjectable pharmaceutical composition may also contain parenteralvehicle(s) (such as sodium chloride and Ringer's dextrose) and/orintravenous vehicle(s) (such as fluid and nutrient replenishers); aswell as other conventional, pharmaceutically acceptable, non-toxicexcipient(s) and additive(s) including salt(s), buffer(s) andpreservative(s) such as antibacterial and/or antifungal agent(s) (e.g.parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like).Prolonged absorption of the injectable composition can be brought aboutby adding agents that can delay absorption (e.g. aluminum monostearateand/or gelatin). The pH and concentration of the various components canreadily be determined by those skilled in the art.

The sterile injectable solution may be prepared by incorporating theactive compound(s), i.e. the molecular imaging agent, and otheringredient(s) in the required amount of an appropriate solvent and thenby sterilizing the resulting mixture, for example, by filtration and/orirradiation.

In general, the dosage of the molecular imaging agent (or pharmaceuticalcomposition comprising thereof) will vary depending on considerationssuch as age, sex and weight of the subject, as well as the particularinflammatory condition suspected to affect the patient, the extent ofthe disease, the tissue(s) of the body to be examined and/or thesensitivity of the imaging label. Factors such as contraindications,therapies, and other variables are also to be taken into account toadjust the dosage of molecular imaging agent to be administered. This,however, can be readily achieved by a trained physician.

In general, one suitable dose of molecular imaging agent, i.e. oflabeled CD31^(shed) ligand, or a pharmaceutical composition comprisingthereof, corresponds to the lowest amount of molecular imaging agent orpharmaceutical composition that is sufficient to allow molecular imagingof any relevant CD31^(shed) present in the subject. To minimize thisdose, it is preferred that administration be intravenous, intramuscular,intraperitoneal or subcutaneous, and preferably proximal to the site tobe examined.

Kits

The present invention also provides a kit comprising material(s) usefulfor carrying out the methods and uses of the invention.

In certain embodiments, the kit comprises at least one CD31^(shed)ligand as above described and at least one imaging label, preferably atleast one radionuclide, and, optionally, instructions for associatingsaid CD31^(shed) ligand and said imaging label, preferably radionuclide,to form a labeled CD31^(shed) ligand according to the invention.

The radionuclide is preferably a short-lived radionuclide, such astechnetium-99m (99mTc), gallium-67 (67Ga), yttrium-90 (90Y), indium-111(111In), rhenium-186 (186Re), and thallium-201 (201TI), more preferably99mTc.

In addition, the kit may further comprise at least one co-ligand, suchas tricine and/or ethylendiaminediacetic acid (EDDA).

In addition, the kit may further comprise one or more of: labellingbuffer, labelling reagent, purification buffer, purification reagent,purification means, injection medium, and/or injection reagent.Protocols for using these buffer(s), reagent(s) and/or means forperforming different steps of the preparation procedure and/oradministration may be included in the kit.

The different components included in the kit may be supplied in a solid(e.g. lyophilized) or liquid form.

The kit may optionally comprise different containers (e.g. vial,ampoule, test tube, flask or bottle) for each individual component. Eachcomponent will generally be suitable as aliquoted in its respectivecontainer or provided in a concentrated form. Other containers suitablefor conducting certain steps of the preparation methods may also beprovided. The individual containers of the kit are preferably maintainedin close confinement for commercial sale.

In certain embodiments, the kit further comprises instructions for usingits components for the imaging of an inflammatory condition as describedherein, and in particular for determining whether a subject suffersfrom, is at risk of having or developing an inflammatory condition or isat risk of recurrence of an inflammatory condition after ananti-inflammatory treatment, as described herein.

Instructions for using the kit according to the invention may compriseinstructions for preparing labeled CD31^(shed) ligand from theCD31^(shed) ligand and the imaging label, instructions concerning dosageand mode of administration of the molecular imaging agent therebyobtained, instructions for performing the detection of CD31^(shed),and/or instructions for interpreting the results obtained. A kit mayalso contain a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts.

The present invention will be further illustrated in view of thefollowing examples and figures.

All references cited herein, including journal articles or abstracts,published or unpublished patent application, issued patents or any otherreferences, are entirely incorporated by reference herein, including alldata, tables, figures and text presented in the cited references.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Radio high performance liquid chromatography (HPLC) of99mTc-HYNIC-P8RI with tricine (A) and tricine/EDDA as coligands (B).HPLC analysis of the acetonitrile fraction shows a major specie (Tr=10.1min) with tricine/EDDA contrary to the multiple species profile withtricine.

FIG. 2: Radio-HPLC stability study of 99mTc-HYNIC-P8RI with tricine/EDDAas coligands. A: plasma stability after 4 h of incubation in humanplasma. B: Radio-HPLC chromatogram of urine collected in a rat bladder 1hour after injection of 74 MBq of 99mTc-HYNIC-P8RI with tricine/EDDA ascoligands. For A and B, peak in the acetonitrile fraction (Tr=10.1 min)corresponds to the initial specie with no signs of degradation.

FIG. 3: SPECT/CT images of [99mTc]EDDA/HYNIC-P8RI in a male wistar ratwith an abdominal aortic aneurysm (AAA) receiving injection ofPorphyromonas gingivalis weekly. Images were acquired 30 min after atracer injection of 74 MBq and 21 days after AAA surgery. The axial (A),coronal (B) and sagittal (C) planes of the abdominal region are shown.It is observed the tracer elimination via kidneys (white arrows in B)and the bladder (white arrow in C). Focal uptake of[99mTc]EDDA/HYNIC-P8RI can be observed at the location of the AAA (blackarrows in A, B, C).

FIG. 4: Comparison of average counts ratio between right hindlimb andleft hindlimb measured in the same region of the thigh fortechnetium-99m radiolabeled mertiatide (n=3) and P8RI injected rats(n=3), in a model of right hindlimb inflammation. Briefly, turpentineoil was injected intramuscularly in Wistar rats right hindlimb and thecontralateral thight was injected similarly and simultaneously withsaline. SPECT/CT acquisitions were performed 48 hours later and 30 minafter injection of 80 MBq of [99mTc]-HYNIC-PEGS-P8RI or[99mTc]-mertiatide. Comparison between the two groups was realized usinga Mann-Withney U test, a p value of less than 0.05 was consideredsignificant.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 corresponds to the sequence of human CD31.

SEQ ID NO: 2 corresponds to the sequence LAPWKK of a 6 amino acidpeptide derived from human or murine CD31.

SEQ ID NO: 3 corresponds to the sequence VRVFLAPWKK of a 10 amino acidpeptide derived from murine CD31, also called PepReg CD31.

SEQ ID NO: 4 corresponds to the sequence VRVILAPWKK of a 10 amino acidpeptide derived from human CD31.

SEQ ID NO: 5 corresponds to the sequence RVFLAPWK of a 8 amino acidpeptide derived from murine CD31, also called P8F.

SEQ ID NO: 6 corresponds to the sequence kwpalfvr of a 8 amino acidpeptide, also called P8RI, having the inverted sequence of SEQ ID NO: 5and consisting of D-amino acids.

SEQ ID NO: 7 corresponds to the sequence RVILAPWK of a 8 amino acidpeptide derived from human CD31.

SEQ ID NO: 8 corresponds to the sequence kwpalivr of a 8 amino acidpeptide having the inverted sequence of SEQ ID NO: 7 and consisting ofD-amino acids.

SEQ ID NO: 9 corresponds to the sequence of murine CD31.

SEQ ID NO: 10 corresponds to the sequence of bovine CD31.

SEQ ID NO: 11 corresponds to the sequence of pig CD31.

SEQ ID NO: 12 corresponds to the amino acids 579 to 601 of sequence SEQID NO: 1.

Sequences SEQ ID NO: 13 to 74 are as defined above.

EXAMPLES Materials and Methods 1. Materials

Reagents were purchased from Sigma-Aldrich Corporation except whenotherwise stated and used as received. P8RI (H-kwpalfvr-OH) wassynthetized on solid phase with a purity>85% analyzed by RP-HPLC/MS.HYNIC- P8RI was prepared by solid phase peptide synthesis using Fmocchemistry (yield after RP-HPLC purification 52%, purity by RP-HPLC 96%).

Na^(99m)TcO₄ ⁻ was obtained from a commercial ⁹⁹Mo/^(99m)Tc generator(TEKCIS®, Iba molecular, France).

2. Analytical Methods 2.1 HPLC

A Dionex Ultimate 3000 system coupled to a Berthold radiometric detectorwas used for RP-HPLC analysis. A ACE 3 C18, 3 μ, 100 Å, 150×4.6 mmcolumn, at a flow rate of 1 mUmin and with UV detection at 220 nm, wasemployed with the following mobile phases, A: 0.1% TFA/water), BAcetonitrile (ACN). Gradient was: 0-2 min 23% B, 2-20 min 23-50% B,20-23 min 50-100% B, 23-25 min 100-23% B, 25-28 min 23% B.

2.2 Thin Layer Chromatography

TLC was performed using a radiochromatogaph (MiniGita, Raytest,Germany). Stationary phase was silica gel (ITLC-SG, Agilenttechnologies, USA) and different mobile phases were employed. MEK wasused to determine the amount of free ⁹⁹mTcO₄ ⁻ (Rf=1), AnticoagulantCitrate Dextrose Solution (ACD-A, Baxter International, USA) todetermine non-peptide-bound ⁹⁹mTc-coligands and ⁹⁹mTcO₄ ⁻ (Rf=1), 60%ACN for ^(99m)Tc-colloid (Rf =0)

3. ^(99m)Tc Radiolabeling 3.1 Tricine as Coligand

In a rubber-sealed N2 purged vial 20 μg of HYNIC- P8RI were incubatedwith 500 μL of a tricine solution (40 mg/mL in PBS 1× buffer pH 7.2), 80μL of a tin(II) solution (1 mg/mL in HCl 0.1N), 1 GBq ^(99m)TcO4- eluateand PBS qs a 3 mL total volume for 30 min at room temperature (RT).

3.2 EDDA as Coligand

In a rubber-sealed N2 purged vial 20 μg of HYNIC- P8RI were incubatedwith 500 μL of a EDDA solution (20 mg/mL in NaOH 0.1N), 80 μL of atin(II) solution (1 mg/mL in HCl 0.1N), 1 GBq ^(99m)TcO4⁻ eluate and PBS1× qs a 3 mL total volume for 30 min at RT.

3.3 Tricine/EDDA Exchange Labeling

Same procedure than tricine as coligand except that 500 μL of a EDDAsolution (20 mg/mL in NaOH 0.1N) was added in the reaction vial andheated for 10 min at 100° C.

4. Purification Procedure

Purification was realized using C18 Sep-Pak cartridge (Sep-Pak C18 PlusLight Cartridge, Waters, USA) preactivated with 10 mL of ethanolfollowed by 10 mL of water and 5 mL of air. After passing theradiolabeling preparation through the cartridge and washing it with 8 mLof water, the radiolabeled peptide was eluted with 80% ACN which wasthen evaporated under vacuum.

5. In Vitro Stability Study

Stability of ^(99m)Tc complexes was assessed in fresh human plasma at37° C. after 0 min, 30 min, 1 hour, 2 hours and 4 hours of incubation ata concentration of 100 pmol/mL. Next, plasma samples were precipitatedwith methanol and centrifuged (20000 g, 10 min). Supernatants werecollected and filtered (Millex-GV 0.22 μm PVDF, Merck Millipore,Germany) then assessed by radio-HPLC.

6. In Vivo Stability Study

74 MBq of 99mTc-HYNIC-P8RI (obtained with tricine/EDDA as coligands) wasinjected to a male wistar rat. After one hour, rat was sacrificed andurine was directly collected from the bladder using a syringe andanalyzed by radio-HPLC after a 0.22 μm filtration (Millex-GV 0.22 μmPVDF, Merck Millipore, Germany).

7. Protein Binding

Protein binding of the purified radiolabeled peptide was determinedafter 0 min, 30 min, 1 hour, 2 hours and 4 hours of incubation in freshhuman plasma at 37° C. and analyzed after size exclusion chromatography(illustra Microspin G-50 Columns, Sephadex G-50, GE Healthcare, UK).G-50 columns were prespun at 2000×g for 1 min then 20 μL of mixture wasadded and the column was centrifuged at 2000×g for 2 min. Proteinbinding of the radiolabeled peptide was estimated by measuring columnsand eluates in a gamma-counter (Cobra II, Packard Bioscience). In thesame time, radiolabeled peptide was incubated for 1 h in PBS 1× at 37°C. as a control.

8. Experimental Models

8.1. Abdominal Aorta Aneurysm (AAA) Model

Rat model of vascular inflammation that has been set up at thelaboratory: the experimental abdominal aorta aneurysm (AAA) induced bylocal infusion of elastase and followed by intravenous injection of aperiodontal bacterium present in human AAA (Porphyromonas gingivalis)and known to induce AAA inflammation (Delbosc et al., 2011, PLoS ONE6(4): e18679. doi:10.1371/journal.pone.0018679).

8.2. Hindlimb Inflammation Model

Rat model of hindlimb inflammation was induced by intramuscularinjection of turpentine oil (150 μl) in the right hindlimb, whereas asaline solution (150 μl) was injected in the left hindlimb(contralateral control).

9. SPECT/CT Imaging—AAA Model

Immediately after intravenous injection via the penis vein of 74 MBq ofradiolabelled HYNIC-P8RI (obtained using Tricine/EDDA), sequentialwhole-body acquisitions (every 10 minutes for the first hour) wereperformed with a hybrid SPECT/CT camera (NanoSPECT/CT, Bioscan Inc.)dedicated to small animals.

10. X ray CT Scanner, SPECT and SPECT/CT Acquisitions—Right HindlimbInflammation

Acquisitions were performed 30 min after injection of 80 MBq of99mTc-HYNIC-P8RI (obtained with tricine/EDDA as coligands) and^(99m)Tc-mertiatide, respectively.

Results 1. Radiolabeling

6-Hydrazinopyridine-3-carboxylic acid (HYNIC) was coupled to theN-terminal amino-acid of the P8RI peptide via a 3(PEG) spacer to obtainHYNIC-P8RI.

HYNIC-P8RI was then labeled at high specific activities (>71 GBq/μmol)using tricine, EDDA or Tricine/EDDA as coligands. Labelling yieldsvaried from 65.5% to 98.3% as shown in Table 2. The tricine/EDDAexchange labeling strategy was chosen because of high labeling yield andresulting in a single major specie as analyzed by HPLC (see FIG. 1).

TABLE 2 Labelling yields of 99mTc-HYNIC-P8RI using different coligandsCo-ligand N yield(%) SD (%) Tricine 3 98.3 0.5 EDDA 3 65.5 7.3Tricine/EDDA 3 93.8 2.8

2. Stability Study

In vitro stability in human plasma revealed a high stability of the99mTc complex with no significant release of radiolabeled impurities orradiolabeled peptide degradation. The RCP (radiochemical purity) wassuperior to 89% after 4 h of incubation (see FIG. 2 A). In vivostability study on rat urine by radio-HPLC analysis showed one majorspecies excreted with a retention time corresponding to the injectedradiotracer. This result indicates that 99mTc-HYNIC-P8RI may be excretedunchanged (see FIG. 28).

3. Protein Binding

Very low levels of protein binding as determined by size exclusionchromatography (<5% after 4 h incubation) were found using theTricine/EDDA coligands exchange labelling strategy. This findingsuggests that 99mTc-HYNIC-P8RI may be a hydrophilic compound.

4. SPECT/CT Imaging—AAA Model

Representative SPECT and CT images were obtained after an acquisitionperformed 30 min after injection of 99mTc-HYNIC-P8RI (obtained withtricine/EDDA as coligands) in AAA rat as shown in FIG. 3. The pattern ofbiodistribution indicated almost exclusive renal uptake and excretion.Interestingly, there was a focal uptake of the radiotracer on the aortapathway corresponding to the location of the AAA.

5. X ray CT Scanner, SPECT and SPECT/CT Acquisitions—Right HindlimbInflammation model

^(99m)Tc- HYNIC-P8RI was further assessed in a rat model of righthindlimb inflammation using turpentine oil.

Mertiatide is a non-specific marker of renal function and was used as anegative control, because of its biodistribution pattern close topeptide P8RI (in particular, low molecular weight and quick clearance).

In this model, the technetium-99m radiolabeled P8RI uptake ratio betweenthe turpentine oil injected hindlimb and the saline injected hindlimbwas higher compared to the control group injected with technetium-99mradiolabeled mertiatide, thereby confirming the specific binding of P8RIto inflammatory sites (see FIG. 4).

Conclusion

The RCP of [99mTc]EDDA/HYNIC-P8RI was >93% (HPLC and ITLC) without anypurification and the specific activity was >71 GBq/pmol. There was nosignificant release of degraded radiolabelled peptide (RCP>89%) and theradiotracer binding to plasma proteins was very low (<5% after 4 hincubation). In vivo, blood clearance of the tracer was almostexclusively renal with a peak activity in kidneys and bladder 1 h afterinjection, corresponding to the unaltered form of the peptide on HPLC.In addition, ^(99m)Tc-HYNIC-P8RI uptake by AAA was detectable from 30min after injection in animals and associated with activated plateletsand leukocytes on immunohistochemistry. ^(99m)Tc-HYNIC-P8RI was alsospecifically detected in inflammatory sites in a model of right hindlimbinflammation. By specifically targeting activated cells involved ininflammation expressing truncated CD31 (CD31^(shed)) and with a rapidblood clearance, radiolabelled P8RI constitutes a useful novel approachin inflammation imaging.

1. A labeled CD31^(shed) ligand comprising a CD31^(shed) ligand and atleast one imaging label.
 2. The labeled CD31^(shed) ligand according toclaim 1, wherein said CD31^(shed) ligand is a peptide, a peptidomimetic,a chemical compound, an antibody, or an aptamer.
 3. The labeledCD31^(shed) ligand according to claim 1, wherein said CD31^(shed) ligandis a single domain antibody or a scFv fragment.
 4. The labeledCD31^(shed) ligand according to claim 1, wherein said CD31^(shed) ligandis a) a peptide selected in the group consisting of: (i) a peptideconsisting of a fragment of 3 to 15 amino acids of the sequence definedby amino acids 579 to 601 of sequence SEQ ID NO: 1, (ii) a peptideconsisting of a fragment of 3 to 15 amino acids of a sequencecorresponding to the amino acids 579 to 601 of sequence SEQ ID NO: 1 ina non-human mammalian CD31, (iii) a peptide of 3 to 15 amino acidsconsisting of a sequence at least 70% identical to the sequence ofpeptide (i), (iv) a peptide consisting of a retro-inverso sequence ofpeptide (i), (ii), or (iii), and (v) the peptide (i), (ii), (iii), or(iv) comprising at least one chemical modification, or b) apeptidomimetic of peptide a).
 5. The labeled CD31^(shed) ligandaccording to claim 1, wherein said CD31^(shed) ligand is a peptideselected in the group consisting of a peptide of sequence SEQ ID NO: 2,a peptide of sequence SEQ ID NO: 3, a peptide of sequence SEQ ID NO: 4,a peptide of sequence SEQ ID NO: 5, a peptide of sequence SEQ ID NO: 6consisting of D-enantiomer amino acids, a peptide of sequence SEQ ID NO:7, and a peptide of sequence SEQ ID NO: 8 consisting of D-enantiomeramino acids.
 6. The labeled CD31^(shed) ligand according to claim 1,wherein said CD31^(shedl) ligand is a peptide of sequence SEQ ID NO: 5,a peptide of sequence SEQ ID NO: 6 consisting of D-enantiomer aminoacids, a peptide of sequence SEQ ID NO: 7, or a peptide of sequence SEQID NO: 8 consisting of D-enantiomer amino acids.
 7. The labeledCD31^(shed) ligand according to claim 1, wherein said imaging label is aradionuclide.
 8. The labeled CD31^(shed) ligand according to claim 1,wherein said imaging label is a radionuclide detectable by PositronEmission Tomography (PET), Single Photon Emission Computed Tomography(SPECT), a hybrid of SPECT and/or PET or their combinations.
 9. Thelabeled CD31^(shed) ligand according to claim 1, wherein said imaginglabel is a radionuclide selected from the group consisting oftechnetium-99m (99mTc), gallium-67 (67Ga), gallium-68 (68Ga) yttrium-90(90Y), indium-111 (111In), rhenium-186 (186Re), fluorine-18 (18F),copper-64 (64Cu), and thallium-201 (201Tl).
 10. The labeled CD31^(shed)ligand according to claim 1, wherein said imaging label is 99mTc. 11.The labeled CD31^(shed) ligand according to claim 1, wherein saidCD31^(shed) ligand is a peptide selected in the group consisting of apeptide of sequence SEQ ID NO: 2, a peptide of sequence SEQ ID NO: 3, apeptide of sequence SEQ ID NO: 4, a peptide of sequence SEQ ID NO: 5, apeptide of sequence SEQ ID NO: 6 consisting of D-enantiomer amino acids,a peptide of sequence SEQ ID NO: 7, and a peptide of sequence SEQ ID NO:8 consisting of D-enantiomer amino acids; and wherein said imaging labelis a radionuclide.
 12. The labeled CD31^(shed) ligand according to claim1, wherein said CD31^(shed) ligand is a peptide of sequence SEQ ID NO: 6consisting of D-enantiomer amino acids or a peptide of sequence SEQ IDNO: 8 consisting of D-enantiomer amino acids; and wherein said imaginglabel is 99mTc.
 13. The labeled CD31^(shed) ligand according to claim 1,wherein said CD31^(shed) ligand comprises6-hydrazinopyridine-3-carboxylic acid (HYNIC).